The Hedgehog (Hh) signaling network regulates embryonic development and adult stem cell maintenance. Altered Hh signaling has been implicated in approximately 25% of all human cancers including those of skin, prostate brain and breast. Smoothened (SMO), a critical component of the Hh pathway, is overexpressed in about 70% of ductal carcinoma in situ (DCIS), and about 30% of invasive breast cancer. In mice, SMO overexpression increases cell division and disrupts mammary gland morphology. SMO also cooperates with the ErbB2 (Her2) oncogene to promote tumor formation in mice. Collectively, these data suggest the importance of Hh signaling in breast cancer. Mechanistically, SMO-driven hyperproliferation does not appear to be completely mediated by the known mechanism of GLI transcription factor activation. Rather, this rapid cell division can be completely blocked by pertussis toxin, a specific inhibitor of "heterotrimeric G- proteins." This finding suggests our hypothesis that SmoM2 activation increases cell proliferation through a unique G-protein mediated mechanism in mammary epithelial cells. To test this hypothesis and to identify critical mediators functioning downstream of the G protein(s), the specific aims are: Specific Aim I: To determine which G1i subunits are expressed in the mammary gland of our SMO mutant mice and to test whether disruption of such subunit(s) function can block the altered mammary gland phenotype in SMO mutant mice using transcript detection &disruption methods coupled with transplantation. Specific Aim II: To test whether SMO-mediated mammary epithelial hyperplasias are a result of 23 subunit signaling through a PTX-sensitive mechanism using protein expression &detection methods. Specific Aim III: To define the molecular mechanism downstream of G-protein activation that drives the SMO- - mediated mammary hyperplastic phenotype in our model using a pharmacological intervention approach. Significance: Since small molecule inhibitors currently under development were designed to inhibit only the GLI-mediated signaling, but not necessarily G protein-mediated signaling, downstream of SMO, the implications of the proposed research on clinical development of Hh signaling inhibitors may be astounding. Training Program: The Lester and Sue Smith Breast Center at Baylor College of Medicine allows training in a collaborative and enriching environment in which basic science, translational, and clinical research are valued equally. This form of training is reinforced by the bi-weekly Research and Development workshops, journal club series, weekly breast cancer seminars and the annual Breast Center retreat. In addition to the training provided by the Breast Center, the department of Molecular and Cellular Biology holds its annual student research symposium, a weekly seminar series and the annual graduate student symposium. All of these events create the perfect environment in which to not only present research to my peers and faculty, but to receive valuable feedback which will only advance my research to a higher level. PUBLIC HEALTH RELEVANCE: The proposed study aims to identify the specific G protein subunits involved in Smoothened (SMO) driven hyperplasias, as well as to identify other critical mediators functioning downstream of the G protein(s). These new targets are important in promoting increased cell proliferation that could be important in the initial stages of breast cancer. Since small molecule inhibitors currently under development were designed to inhibit only the GLI-mediated signaling, but not necessarily G protein-mediated signaling, downstream of SMO, the implications of the proposed research on clinical development of Hedgehog signaling inhibitors may be astonishing.